We have identified a group of neurons in the arcuate nucleus of the sheep that co-express two neuropeptides: the endogenous opioid peptide, dynorphin A (DYN), and the tachykinin, neurokinin B (NKB). There is strong evidence that DYN mediates the negative feedback actions of progesterone or gonadotropin-releasing hormone (GnRH) and indirect evidence for a similar role for NKB. We have also recently demonstrated that many of these neurons also contain kisspeptin, an important newly discovered stimulator of GnRH. Specifically over 80% of the DYN-NKB neurons in the caudal arcuate also contain kisspeptin, but that this falls to less than 50% in the rostral arcuate. Although all three peptides are found in the same neurons that appear to mediate steroid negative feedback, they have different effects on GnRH neurons: DYN inhibits GnRH secretion, while kisspeptin and most likely NKB, stimulate it. Therefore, we postulate that the negative feedback actions of ovarian steroids have differential effects on expression and release of these three neuropeptides, stimulating DYN and inhibiting Kisspeptin and NKB. In this proposal, we will test this hypothesis at three different levels: 1) integrative release into the CSF, 2) regionally within the arcuate nucleus, and 3) at the single cell level in the caudal and rostral portions of the arcuate. First we use liquid chromoatograph and mass spectrometry to monitor all three peptides in CSF collected from luteal phase, ovariectomized and steroid-treated ovariectomized ewes. We will then collect hypothalamic tissue from these ewes and cut alternating thick (100 Cm) and thin (20 mm) sections with a cryostat. The region containing the arcuate nucleus will be microdissected from the thick sections and pooled into rostral, middle, and caudal regions. The right side pools will be used to measure mRNA levels of the appropriate prepropeptide by RT-PCR and the left side pools used to monitor protein concentrations be Western analysis. Finally, the thin frozen sections will be used for either single cell RT-PCR or dual in situ hybridization to determine if the differential actions of ovarian steroids occur in the same neurons. The results of this study will provide novel information on control of three transmitters that have been implicated in the negative feedback actions of steroids in both sheep and women. It is also clear that abnormalities in steroid negative feedback occur in a number of pathologies, including polycystic ovarian syndrome (PCOS) and anorexia nervosa. For example, the increased episodic LH secretion in PCOS is due, in part, to decreased response to progesterone negative feedback. Thus, these studies may provide information important for the development of better treatments of these pathological disruptions of reproductive function. [unreadable] [unreadable] [unreadable]